Arthropod toxins inhibiting Ca2+ and Na+ channels prevent AC‐1001 H3 peptide‐induced apoptosis

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca²⁺ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U₅‐scytotoxin‐Sth1a, ω‐hexatoxin‐Hv1a, ω‐theraphotoxin‐Hhn2a, and μ‐agatoxin‐Aa1a toxins—inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC‐1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO‐K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC‐1001 H3 and reduce the level of natural apoptosis in CHO‐K1 cells. Cell incubation with apoptosis inducer AC‐1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms.     

Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.     

Improving coiled-coil stability by optimizing ionic interactions

Alpha-helical coiled coils are a common protein oligomerization motif stabilized mainly by hydrophobic interactions occurring along the coiled-coil interface. We have recently designed and solved the structure of a two-heptad repeat coiled-coil peptide that is stabilized further by a complex network of inter- and intrahelical salt-bridges in addition to the hydrophobic interactions. Here, we extend and improve the de novo design of this two heptad-repeat peptide by four newly designed peptides characterized by different types of ionic interactions. The contribution of these different types of ionic interactions to coiled-coil stability are analyzed by CD spectroscopy and analytical ultracentrifugation. We show that all peptides are highly alpha-helical and two of them are 100% dimeric under physiological conditions. Furthermore, we have solved the X-ray structure of the most stable of these peptides and the rational design principles are verified by comparing this structure to the structure of the parent peptide. We show that by combining the most favorable inter- and intrahelical salt-bridge arrangements it is possible to design coiled-coil oligomerization domains with improved stability properties.     

Desmoglein versus non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens

Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces "desmosomal" signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by approximately 45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by approximately 50% and TUNEL positivity by approximately 25%. The p38 MAPK inhibitor PD169316 inhibited these effects by approximately 15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked approximately 50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies.     

A soluble C1b protein and its regulation of soluble type 7 adenylyl cyclase

Adenylyl cyclase (AC) is a prototypical cell-signaling molecule expressed in virtually all organisms from bacteria to man. While C1b, a poorly conserved region within mammalian AC, has been implicated in numerous isoform-specific regulatory properties, no one has purified the C1b region as a functional protein to homogeneity in order to study its role in enzyme function. We hypothesize that C1b is an internal regulatory subunit. To pursue this hypothesis, we constructed several soluble C1b proteins from type VII AC, arriving at one, 7C1b-S, which can be expressed and purified from Escherichia coli. 7C1b-S is relatively stable, as demonstrated by limited proteolytic analysis, circular dichroism, and UV Raman spectroscopy. Using size-exclusion chromatography and co-immunoprecipitation we demonstrate that 7C1b-S interacts with a cardinal activator of AC (Gsalpha) and with the conserved first catalytic domain (C1a) of type VII AC. We show that 7C1b-S inhibits Gsalpha-stimulated and Gsalpha-forskolin stimulated activity in our soluble ACVII model system. On the basis of these results, we suggest that 7C1b-S meets basic criteria to serve as a model protein for the C1b region and may be used as a prototype to develop other isoform C1b soluble model proteins to further investigate the role of this domain in isoform-specific regulation of adenylyl cyclase.